RECOMBINANT BIOTIN CARBOXYLASE DOMAINS FOR IDENTIFICATION OF ACETYL CoA CARBOXYLASE INHIBITORS

ABSTRACT

A peptide comprising an Acetyl CoA carboxylase (ACCase) having a deleted biotin binding domain, having a deleted carboxy transferase domain, and having a functional biotin carboxylase (BC) domain is described. A nucleic acid that encodes the peptide described above and a recombinant host cell that contains the nucleic acid and expresses the encoded peptide is also described. A method of identifying Acetyl CoA carboxylase inhibitors, fungicides, herbicides and pharmaceuticals is also described herein.

RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 10/633,835, filed Aug. 4, 2003, which claims thebenefit, under 35 U.S.C. 119(e), of U.S. Provisional Patent ApplicationNo. 60/401,170, filed Aug. 5, 2002, the contents of which are hereinincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to a peptide comprising a biotincarboxylase domain and fragments thereof useful for the identificationof Acetyl CoA carboxylase inhibitors, which in turn are useful amongother things as fungicides, insecticides, nematicides, herbicides andpharmaceuticals.

BACKGROUND OF THE INVENTION

Acetyl CoA carboxylase (ACCase) catalyzes the first committed step infatty acid biosynthesis and has also been chemically validated as anherbicide and fungicide target. Structurally, ACCases are biotinylated,multifunctional enzymes comprised of three domains: a biotin carboxylasedomain, a biotin binding site, and a carboxytransferase domain. Inprokaryotic ACCases, as well as in the plastidic isoforms of most plantACCases, the three domains reside on three distinct, dissociableproteins. In contrast, in most eukaryotic ACCases the three domainsreside on a single polypeptide of 160 kD to 280 kD. In their nativestate, the eukaryotic enzymes are typically dimers or tetramers rangingin size from approximately 400-800 kD.

The ACCase reaction takes place at two catalytic sites via two partialreactions: the ATP dependent carboxylation of the enzyme-bound biotinprosthetic group, and the subsequent transfer of the carboxyl group frombiotin to acetyl CoA to form malonyl CoA. The natural product soraphenhas been demonstrated to be a broad-spectrum fungicide that acts byinhibiting the biotin carboxylase reaction of ACCase. ACCase's are knownto be low abundant and labile proteins. These properties impede theidentification of new ACCase inhibitors.

The present invention provides a peptide comprising a biotin carboxylasedomain and fragments thereof useful for the identification of Acetyl CoAcarboxylase inhibitors, which in turn are useful among other things asfungicides, insecticides, nematicides, herbicides and pharmaceuticals.

SUMMARY OF THE INVENTION

According to embodiments of the present invention, the present inventionrelates to a peptide comprising an Acetyl CoA carboxylase (ACCase)having a deleted biotin binding domain, having a deleted carboxytransferase domain, and having a functional biotin carboxylase (BC)domain (e.g., capable of binding soraphen). In some embodiments of theinvention where such BC domains are used as counterselection agents inconjunction with peptides or BC domains as described above, the peptideor BC domain is non-functional.

“Functional” as used herein refers to a BC domain that binds soraphenwith similar affinity as enzymatically active, full length ACCaseprotein. Thus, “non-functional” as used herein refers to a BC domainthat does not bind soraphen. These non-functional BC domains would befunctional with respect to enzyme activity/catalytic function whenincorporated into an intact ACCase.

The carboxylase (and corresponding peptide) may be from any suitablesource, including plant, animal (e.g., mammalian), insect, yeast, andfungal carboxylases/peptides.

According to other embodiments of the present invention, the carboxylase(and corresponding peptide) is from Ustilago maydis carboxylase.

According to still other embodiments of the present invention, thecarboxylase (and corresponding peptide) is from Phytophthora infestanscarboxylase.

According to still other embodiments of the present invention, thecarboxylase (and corresponding peptide) are from Magnaporthe grisea,Saccharomyces cerevisiae and Homo sapiens.

According to other embodiments of the present invention, the presentinvention relates to the molecules described above wherein therespective peptides are each an Acetyl CoA carboxylase (ACCase) having adeleted biotin binding domain, having a deleted carboxy transferasedomain, and having a functional biotin carboxylase domain comprisingamino acids as detailed in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, or 16,and functional fragments thereof.

According to other embodiments of the present invention, the moleculesdescribed above are each a monomer.

According to still other embodiments of the present invention, thepresent invention relates to the molecules described above wherein therespective carboxylase domains bind to compounds that modulate AcetylCoA carboxylase activity.

According to other embodiments of the present invention, the carboxylasedomains bind to competitive inhibitors, noncompetitive inhibitors, andalso binds to soraphen.

According to other embodiments of the present invention, the presentinvention relates to a nucleic acid that encodes a peptide comprising anAcetyl CoA carboxylase (ACCase) having a deleted biotin binding domain,having a deleted carboxy transferase domain, and having a functionalbiotin carboxylase domain, such as described above and furtherhereinbelow.

According to other embodiments of the present invention, the presentinvention relates to a recombinant host cell that contains a nucleicacid as described above and expresses the encoded peptide.

According to other embodiments of the present invention, the presentinvention relates to a method of identifying Acetyl CoA carboxylaseinhibitors, or activators, comprising a) combining a peptide asdescribed above and a compound to be tested for the ability to bind tosaid biotin carboxylase domain, under conditions that permit binding tosaid biotin carboxylase domain, and b) determining whether or not saidcompound binds to said biotin carboxylase domain, the presence ofbinding indicating said compound is or may be an Acetyl CoA carboxylaseinhibitor. Such compounds are candidates for and useful as pesticides,including but not limited to insecticides, nematocides, fungicides,and/or herbicides, and/or also pharmaceuticals, including but notlimited to antifungals.

According to other embodiments of the present invention, the presentinvention relates to a method of identifying Acetyl CoA carboxylaseinhibitors, further comprising the steps of c) employing a compoundidentified as binding in step (b) in an assay to detect inhibition ofAcetyl CoA carboxylase activity; and d) selecting a compound identifiedin step (c) that inhibits Acetyl CoA carboxylase activity.

According to still other embodiments of the present invention, thepresent invention relates to a method of identifying fungicides,comprising a) combining a peptide as described above and a compound tobe tested for the ability to bind to said biotin carboxylase domain,under conditions that permit binding to said biotin carboxylase domain,b) determining whether or not said compound binds to said biotincarboxylase domain, the presence of binding indicating said compound isor may be a fungicide, c) employing a compound identified as binding instep (b) in an assay to detect inhibition of Acetyl CoA carboxylaseactivity, and d) selecting a compound identified in step (c) thatinhibits Acetyl CoA carboxylase activity.

According to still other embodiments of the present invention, thepresent invention relates to the use of a peptide or compound asdescribed above for carrying out a method as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates full-length ACCase protein from Ustilago maydis(pCS11) with the three functional domains detailed.

FIG. 2 illustrates soraphen binding to and inhibition of the full-lengthpCS11 protein.

FIG. 3 illustrates soraphen binding to the Ustilago BC domain (pCS8)with comparable affinity to full length ACCase (pCS11).

FIG. 4 illustrates soraphen binding to Phytopthora infestans BC domain.

FIG. 5 illustrates the biotin carboxylase domain of Ustilago peptide(pCS8) compared to full-length Ustilago ACCase.

FIG. 6 illustrates the amino acid sequence of Ustilago maydis ACCase BCdomain, amino acids 2-560 (pCS8, SEQ ID NO: 2)(Taken from Full LengthAmino Acid Sequence for Ustilago maydis ACCase, Accession Number:Z46886; A. Bailey, J. Keon, J. Owen, and J. Hargreaves, ACC1 gene,encoding acetyl-CoA carboxylase, is essential for growth in Ustilagomaydis, Mol. Gen. Genet. 249 (2), 191-201 (1995)).

FIG. 7 illustrates the amino acid sequence of Phytopthora infestansACCase BC domain, amino acids 1-555 (pCS15, SEQ ID NO: 4).

FIG. 8 illustrates anion Exchange Chromatography of pCS8 peptide.

FIG. 9A illustrates spectrophotometric assay absorbance traces for E.coli BC and FIG. 9B shows (i) spectrophotometric and (ii) 14C isotopeexchange activity assays on pCS11 protein.

FIG. 10 illustrates the alignment of Ustilago (SEQ ID NO: 2),Phytophthora (SEQ ID NO: 4), Magnaporthe (SEQ ID NO: 6) and yeast (SEQID NO: 8) ACCase BC domains.

FIG. 11 illustrates soraphen binding to the Magnaporthe BC domain.

FIG. 12 illustrates soraphen binding to the human ACC1 BC domain.

FIG. 13 illustrates the alignment of the human ACC1 BC (SEQ ID NO: 10)and ACC2 BC (SEQ ID NO: 12) domains with the Ustilago ACCase BC (SEQ IDNO: 2) domain.

FIG. 14 illustrates dissociation experiments using [³H]-soraphen todetermine the soraphen off rate for Ustilago ACCase BC domain.

FIG. 15 illustrates the binding of [³H]-soraphen A and soraphen Cconjugates to (A) the Ustilago ACCase BC domain and (B) the full-lengthUstilago ACCase protein.

FIG. 16 depicts soraphen binding to wild type and mutant S. cerevisiaeACCase BC domain peptides.

FIG. 17 illustrates soraphen A binding to wild-type and mutantfull-length S. cerevisiae ACCase.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will now be described more fully hereinafter withreference to the accompanying figures, in which preferred embodiments ofthe invention are shown. This invention may, however, be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art. Thedisclosures of all United States patent references cited herein are tobe incorporated by reference herein in their entirety.

Described herein is the use of recombinant, isolated, biotin carboxylasedomains for the discovery of new Acetyl CoA carboxylase (ACCase)inhibitors. A biotin carboxylase (BC) domain from the ACCase gene of thebasidiomycete Ustilago maydis was isolated, cloned, expressed, andcharacterized. The isolated BC domain was shown to have similarhigh-affinity, soraphen-binding properties as the full-length protein.In contrast to the full-length protein (FIG. 1), however, the BC domainis significantly smaller and can be expressed at higher levels, is morestable, and exists as a monomer. The isolated BC domain is useful forscreening new ACCase inhibitors. The BC domain from the oomycetePhytophthora infestans was also cloned. A full-length ACCase sequencefrom this organism has not been published. The appropriate fragment wascloned utilizing PCR using primers derived from published EST's thatshowed homology to sequences flanking the soraphen-binding domain thatwas identified in the Ustilago gene. The recombinantly expressedPhytopthora BC domain exhibited high-affinity soraphen-binding. BCdomains from M. grisea, S. cerevisiae, and H. sapiens were alsosimilarly cloned and determined to exhibit high-affinitysoraphen-binding, thus demonstrating the applicability of this approachto distantly related organisms.

The terminology used in the description of the invention herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the invention. As used in the description ofthe invention and the appended claims, the singular forms “a”, “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise.

As used herein, an “isolated” nucleic acid (e.g., an “isolated DNA” oran “isolated genomic RNA”) means a nucleic acid separated orsubstantially free from at least some of the other components of thenaturally occurring organism or virus, for example, the cell or viralstructural components or other polypeptides or nucleic acids commonlyfound associated with the nucleic acid.

Likewise, an “isolated” polypeptide means a polypeptide that isseparated or substantially free from at least some of the othercomponents of the naturally occurring organism or virus, for example,the cell or viral structural components or other polypeptides or nucleicacids commonly found associated with the polypeptide. As used herein,the terms “polypeptide” and “peptide” have the same meaning.

As used herein, the terms “deleted” or “deletion” mean either totaldeletion of the specified segment or the deletion of a sufficientportion of the specified segment to render the segment inoperative ornonfunctional (e.g., does not encode a functional peptide, whereinfunctional is defined as the ability to bind soraphen), in accordancewith common usage. See, e.g. U.S. Pat. No. 6,180,362; U.S. Pat. No.5,689,039.

As used herein, the term modulation of Acetyl CoA carboxylase activityrefers to the ability of a compound to alter the activity of the enzyme.The alteration may be by enhancing or decreasing the activity of theenzyme, or by causing the enzyme to function in a manner other than thatobserved in the absence of the compound.

Also as used herein, the term activator refers to the ability of acompound to initiate and/or enhance Acetyl CoA carboxylase activity(e.g., an agonist).

The term inhibitor as used herein refers to the ability of a compound todecrease and/or terminate Acetyl CoA carboxylase activity (e.g., anantagonist).

As used herein, test compounds refer to compounds that may bind thebiotin carboxylase domain, under conditions that permit binding to thebiotin carboxylase domain. The presence of binding indicating thecompound is or may be an Acetyl CoA carboxylase inhibitor. Moreover,binding of the compound to the biotin carboxylase domain may indicatethat the compound may be a fungicide, insecticide, nematicide, orherbicide or may be a pharmaceutical (e.g., a compound that reduces,controls, inhibits or otherwise regulates weight gain in a human oranimal subject, particularly compounds that are inhibitors of human ormammalian ACC2, and more particularly compounds that preferentiallyinhibit or antagonize human or mammalian ACC2 and not human or mammalianACC1, see, e.g., L. Abu-Elheiga et al., Science 291, 2613 (30 Mar.2001)). Additionally, binding of the test compound refers to specificbinding wherein the binding interaction between the BC domain and testcompounds is high. The dissociation constant of the BC domain complexesis from about 10⁻⁴ M to about 10⁻¹⁴ M, more preferably 10⁻⁷ to 10⁻¹⁴,and still more preferably 10⁻⁸ to 10⁻¹⁴ and most preferably lower than2×10⁻⁹ M. The test compound may be identified by any available means,including but not limited to the Evolutionary Chemistry processdescribed herein below.

Amino acid sequences disclosed herein are presented in the amino tocarboxy direction, from left to right. The amino and carboxy groups arenot presented in the sequence. Nucleotide sequences are presented hereinby single strand only, in the 5′ to 3′ direction, from left to right.Nucleotides and amino acids are represented herein in the mannerrecommended by the IUPAC-IUB Biochemical Nomenclature Commission, or(for amino acids) by three-letter code, in accordance with 37 C.F.R§1.822 and established usage. See, e.g., Patent In User Manual, 99-102(November 1990) (U.S. Patent and Trademark Office).

In general, the term “peptide” refers to a molecular chain of aminoacids with a biological activity (e.g., capacity to bind soraphen). Ifrequired, it can be modified in vivo and/or in vitro, for example byglycosylation, myristoylation, amidation, carboxylation orphosphorylation; thus inter alia oligopeptides and polypeptides areincluded. It is understood however that the peptides of the presentinvention do not extend to native proteins which may contain thedisclosed peptides. The peptides disclosed herein may be obtained, forexample, by synthetic or recombinant techniques known in the art. Itwill also be understood that amino acid and nucleic acid sequences mayinclude or exclude additional residues, such as additions or deletionsof N- or C-terminal amino acids or 5′ or 3′ sequences, and yet still beessentially as set forth as disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological activity (e.g., capacity to bind soraphen). Thus, up to about10, 20, 30, or about 40 amino acids may be deleted from either, or both,the N- and/or C-terminus of the peptide, so long as a functional biotincarboxylase domain (e.g., soraphen binding) is retained. Examples ofsuch peptides are peptides having the amino acid sequence given in SEQID NO: 14, 16 and 17 through 71 herein. Note that, for BC domains ofhuman ACC1 and ACC2, up to 102 and 244 amino acids, respectively may bedeleted from the N-terminal end, alone or in combination with the abovelisted C-terminal deletions, so long as a function biotin carboxylasedomain (e.g., soraphen binding) is retained.

“Nucleic acid sequence” as used herein refers to an oligonucleotide,nucleotide, or polynucleotide, and to DNA or RNA of genomic or syntheticorigin which may be single- or double-stranded, and represent the senseor antisense strand. Suitable nucleic acid sequences encoding an ACCasebiotin carboxylase (BC) domain (that is, an ACCase having a deletedbiotin binding domain and a deleted carboxy transferase domain) include,for example, a nucleic acid encoding a Ustilago maydis BC domain.Examples of such are given as SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, and 15herein.

Polynucleotides of the present invention include those coding forpeptides homologous to, and having essentially the same biologicalproperties as, the peptides disclosed herein. For example, the DNAsequences disclosed herein as SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, and 15.This definition is intended to encompass natural allelic sequencesthereof. Thus, isolated DNA or cloned genes of the present invention canbe of any species of origin. Thus, polynucleotides that hybridize to anyone or more of the DNA sequences disclosed herein as SEQ ID NOS: 1, 3,5, 7, 9, 11, 13, and 15 and which code on expression for an ACCase BCdomain, are also an aspect of the invention. Conditions which willpermit other polynucleotides that code on expression for a protein orpeptide of the present invention to hybridize to the DNA of SEQ IDNOS:1, 3, 5, 7, 9, 11, 13, and 15 herein can be determined in accordancewith known techniques.

For example, hybridization of such sequences may be carried out underconditions of reduced stringency, medium stringency or even stringentconditions (e.g., conditions represented by a wash stringency of 35-40%Formamide with 5×Denhardt's solution, 0.5% SDS and 1×SSPE at 37° C.;conditions represented by a wash stringency of 40-45% Formamide with5×Denhardt's solution, 0.5% SDS, and 1×SSPE at 42° C.; and conditionsrepresented by a wash stringency of 50% Formamide with 5×Denhardt'ssolution, 0.5% SDS and 1×SSPE at 42° C., respectively) to DNA of SEQ IDNOS:1, 3, 5, 7, 9, 11, 13, and 15 herein in a standard hybridizationassay. See, e.g., J. Sambrook et al., Molecular Cloning, A LaboratoryManual (2d Ed. 1989) (Cold Spring Harbor Laboratory). In general,sequences which code for proteins or peptides of the present inventionand which hybridize to the DNA of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, and15, for example, will be at least 60% or 75% identical or homologous,85% identical or homologous, 90% identical or homologous and even 95%identical or homologous, or more with one or more of SEQ ID NOS:1, 3, 5,7, 9, 11, 13, and 15.

Mathematical algorithms can be used to determine the percent identity oftwo sequences. Non-limiting examples of mathematical algorithms are thealgorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5877; the algorithm of Myers and Miller (1988) CABIOS4:11-17; the local homology algorithm of Smith et al. (1981) Adv. Appl.Math. 2:482; the homology alignment algorithm of Needleman and Wunsch(1970) J. Mol. Biol. 48:443-453; and the search-for-similarity-method ofPearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448.

Various computer implementations based on these mathematical algorithmshave been designed to enable the determination of sequence identity. TheBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403 are basedon the algorithm of Karlin and Altschul (1990) supra. Searches to obtainnucleotide sequences that are homologous to nucleotide sequences of thepresent invention can be performed with the BLASTN program, score=100,wordlength=12. To obtain amino acid sequences homologous to sequencesencoding a protein or polypeptide of the current invention, the BLASTXprogram may be used, score=50, wordlength=3. Gapped alignments may beobtained by using Gapped BLAST as described in Altschul et al. (1997)Nucleic Acids Res. 25:3389. To detect distant relationships betweenmolecules, PSI-BLAST can be used. See Altschul et al. (1997) supra. Forall of the BLAST programs, the default parameters of the respectiveprograms can be used.

Further, polynucleotides that code for proteins or peptides of thepresent invention, or polynucleotides that hybridize to that as SEQ IDNO:1, 3, 5, 7, 9, 11, 13, and 15, or polynucleotides having sequenceidentity or homology thereto as described above, for example, but whichdiffer in codon sequence therefrom due to the degeneracy of the geneticcode, are also an aspect of this invention. The degeneracy of thegenetic code, which allows different nucleic acid sequences to code forthe same protein or peptide, is well known in the literature. See, e.g.,U.S. Pat. No. 4,757,006 to Toole et al. at Col. 2, Table 1.

The production of cloned genes, recombinant DNA, vectors, transformedhost cells, proteins and protein fragments by genetic engineering iswell known. See, e.g., U.S. Pat. No. 4,761,371 to Bell et al. at Col. 6line 3 to Col. 9 line 65; U.S. Pat. No. 4,877,729 to Clark et al. atCol. 4 line 38 to Col. 7 line 6; U.S. Pat. No. 4,912,038 to Schilling atCol. 3 line 26 to Col. 14 line 12; and U.S. Pat. No. 4,879,224 toWallner at Col. 6 line 8 to Col. 8 line 59.

PCR is the polymerase chain reaction—a technique for copying thecomplementary strands of a target DNA molecule simultaneously for aseries of cycles until the desired amount is obtained. First, primersare synthesized that have nucleotide sequences complementary to the DNAthat flanks the target region. The DNA is heated to separate thecomplementary strands and then cooled to let the primers bind to theflanking sequences. A heat-stable DNA polymerase is added, and thereaction is allowed to proceed for a series of replication cycles.Twenty will yield a millionfold amplification; thirty cycles will yieldan amplification factor of one billion.

A vector is a replicable DNA construct. Vectors are used herein eitherto amplify DNA encoding the proteins or peptides of the presentinvention or to express the proteins or peptides of the presentinvention. An expression vector is a replicable DNA construct in which aDNA sequence encoding the proteins of the present invention is operablylinked to suitable control sequences capable of effecting the expressionof proteins or peptides of the present invention in a suitable host. Theneed for such control sequences will vary depending upon the hostselected and the transformation method chosen. Generally, controlsequences include a transcriptional promoter, an optional operatorsequence to control transcription, a sequence encoding suitable mRNAribosomal binding sites, and sequences which control the termination oftranscription and translation. Amplification vectors do not requireexpression control domains. All that is needed is the ability toreplicate in a host, usually conferred by an origin of replication, anda selection gene to facilitate recognition of transformants.

Vectors comprise plasmids, viruses (e.g., adenovirus, cytomegalovirus),phage, retroviruses and integratable DNA fragments (i.e., fragmentsintegratable into the host genome by recombination). The vectorreplicates and functions independently of the host genome, or may, insome instances, integrate into the genome itself. Expression vectorsshould contain a promoter and RNA binding sites which are operablylinked to the gene to be expressed and are operable in the hostorganism.

DNA regions are operably linked or operably associated when they arefunctionally related to each other. For example, a promoter is operablylinked to a coding sequence if it controls the transcription of thesequence; a ribosome binding site is operably linked to a codingsequence if it is positioned so as to permit translation. Generally,operably linked means contiguous and, in the case of leader sequences,contiguous and in reading phase.

Transformed host cells are cells which have been transformed ortransfected with vectors containing DNA coding for proteins or peptidesof the present invention and need not express protein or peptide.However, in the present invention, the cells preferably express theprotein or peptide.

Suitable host cells include prokaryotes, yeast cells, or highereukaryotic organism cells. Prokaryote host cells include gram negativeor gram positive organisms, for example Escherichia coli (E. coli) orBacilli. Higher eukaryotic cells include established cell lines ofmammalian origin as described below. Exemplary host cells are E. coliW3110 (ATCC 27,325), E. coli B, E. coli X1776 (ATCC 31,537), E. coli 294(ATCC 31,446). A broad variety of suitable prokaryotic and microbialvectors are available. E. coli is typically transformed using pBR322.See Bolivar et al., Gene 2, 95 (1977). Promoters most commonly used inrecombinant microbial expression vectors include the beta-lactamase(penicillinase) and lactose promoter systems (Chang et al., Nature 275,615 (1978); and Goeddel et al., Nature 281, 544 (1979), a tryptophan(trp) promoter system (Goeddel et al., Nucleic Acids Res. 8, 4057 (1980)and EPO App. Publ. No. 36,776) and the tac promoter (H. De Boer et al.,Proc. Natl. Acad. Sci. USA 80, 21 (1983). The promoter andShine-Dalgarno sequence (for prokaryotic host expression) are operablylinked to the DNA of the present invention, i.e., they are positioned soas to promote transcription of the messenger RNA from the DNA.

Expression vectors should contain a promoter which is recognized by thehost organism. This generally means a promoter obtained from theintended host. Promoters most commonly used in recombinant microbialexpression vectors include the beta-lactamase (penicillinase) andlactose promoter systems (Chang et al., Nature 275, 615 (1978); andGoeddel et al., Nature 281, 544 (1979), a tryptophan (trp) promotersystem (Goeddel et al., Nucleic Acids Res. 8, 4057 (1980) and EPO App.Publ. No. 36,776) and the tac promoter (H. De Boer et al., Proc. Natl.Acad. Sci. USA 80, 21 (1983). While these are commonly used, othermicrobial promoters are suitable. Details concerning nucleotidesequences of many have been published, enabling a skilled worker tooperably ligate them to DNA encoding the protein in plasmid or viralvectors (Siebenlist et al., Cell 20, 269 (1980). The promoter andShine-Dalgarno sequence (for prokaryotic host expression) are operablylinked to the DNA encoding the desired protein, i.e., they arepositioned so as to promote transcription of the protein messenger RNAfrom the DNA.

Eukaryotic microbes such as yeast cultures may be transformed withsuitable protein-encoding vectors. See e.g., U.S. Pat. No. 4,745,057.Saccharomyces cerevisiae is the most commonly used among lowereukaryotic host microorganisms, although a number of other strains arecommonly available. Yeast vectors may contain an origin of replicationfrom the 2 micron yeast plasmid or an autonomously replicating sequence(ARS), a promoter, DNA encoding the desired protein, sequences forpolyadenylation and transcription termination, and a selection gene. Anexemplary plasmid is YRp7, (Stinchcomb et al., Nature 282, 39 (1979);Kingsman et al., Gene 7, 141 (1979); Tschemper et al., Gene 10, 157(1980). This plasmid contains the trp1 gene, which provides a selectionmarker for a mutant strain of yeast lacking the ability to grow intryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85, 12(1977). The presence of the trp1 lesion in the yeast host cell genomethen provides an effective environment for detecting transformation bygrowth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters formetallothionein, 3-phospho-glycerate kinase (Hitzeman et al., J. Biol.Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Reg. 7, 149 (1968); and Holland et al., Biochemistry 17, 4900(1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Suitable vectors and promoters for use in yeast expressionare further described in R. Hitzeman et al., EPO Publn. No. 73,657.

Cultures of cells derived from multicellular organisms are a desirablehost for recombinant protein synthesis. In principal, any highereukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture, including insect cells. Propagation of such cellsin cell culture has become a routine procedure. See Tissue Culture,Academic Press, Kruse and Patterson, editors (1973). Examples of usefulhost cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO)cell lines, and W1138, BHK, COS-7, CV, and MDCK cell lines. Expressionvectors for such cells ordinarily include (if necessary) an origin ofreplication, a promoter located upstream from the gene to be expressed,along with a ribosome binding site, RNA splice site (ifintron-containing genomic DNA is used), a polyadenylation site, and atranscriptional termination sequence.

The transcriptional and translational control sequences in expressionvectors to be used in transforming vertebrate cells are often providedby viral sources. For example, commonly used promoters are derived frompolyoma, Adenovirus 2, and Simian Virus 40 (SV40). See, e.g., U.S. Pat.No. 4,599,308. The early and late promoters are useful because both areobtained easily from the virus as a fragment which also contains theSV40 viral origin of replication. See Fiers et al., Nature 273, 113(1978). Further, the protein promoter, control and/or signal sequences,may also be used, provided such control sequences are compatible withthe host cell chosen.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral source (e.g. Polyoma, Adenovirus, VSV, or BPV), or may beprovided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter may besufficient.

Host cells such as insect cells (e.g., cultured Spodoptera frugiperdacells) and expression vectors such as the baculorivus expression vector(e.g., vectors derived from Autographa californica MNPV, Trichoplusia niMNPV, Rachiplusia ou MNPV, or Galleria ou MNPV) may be employed to makeproteins useful in carrying out the present invention, as described inU.S. Pat. Nos. 4,745,051 and 4,879,236 to Smith et al. In general, abaculovirus expression vector comprises a baculovirus genome containingthe gene to be expressed inserted into the polyhedrin gene at a positionranging from the polyhedrin transcriptional start signal to the ATGstart site and under the transcriptional control of a baculoviruspolyhedrin promoter.

Host cells transformed with nucleotide sequences encoding a protein orpeptide of the invention may be cultured under conditions suitable forthe expression and recovery of the protein from cell culture. Theprotein produced by a transformed cell may be secreted or containedintracellularly depending on the sequence and/or the vector used. Aswill be understood by those of skill in the art, expression vectorscontaining polynucleotides which encode a protein or peptide of theinvention may be designed to contain signal sequences which directsecretion of the protein or peptide through a prokaryotic or eukaryoticcell membrane. Other constructions may be used to join sequencesencoding the protein or peptide to nucleotide sequence encoding apolypeptide domain which will facilitate purification of solubleproteins. Such purification facilitating domains include, but are notlimited to, metal chelating peptides such as histidine-tryptophanmodules that allow purification on immobilized metals, protein A domainsthat allow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp., Seattle, Wash.). The inclusion of cleavable linker sequences suchas those specific for Factor XA or enterokinase (Invitrogen, San Diego,Calif.) between the purification domain and the protein or peptide ofthe invention may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containinga protein or peptide of the invention and a nucleic acid encoding 6histidine residues preceding a thioredoxin or an enterokinase cleavagesite. The histidine residues facilitate purification on IMAC(immobilized metal ion affinity chromatography) as described in Porath,J. et al. (1992, Prot. Exp. Purif. 3: 263-281) while the enterokinasecleavage site provides a means for purifying the protein or peptide ofthe invention from the fusion protein. A discussion of vectors whichcontain fusion proteins is provided in Kroll, D. J. et al. (1993; DNACell Biol. 12:441-453).

ACCase activity can be measured spectrophotometrically and also throughthe isotope exchange technique. ACCase activity was measuredspectrophotometrically by coupling the production of ADP to theoxidation of NADH using pyruvate kinase and lactate dehydrogenase. Thisassay was used to measure overall ACCase activity by supplying acetylCoA as a substrate. Activity of the full length Ustilago ACCase isdetailed in FIG. 2. ACCase activity was measured by way of isotopeexchange based upon the fact that ACCase catalyzes the formation ofmalonyl CoA from acetyl-CoA and bicarbonate. The isotope exchange assayis designed to monitor the incorporation of ¹⁴C from bicarbonate intothe malonyl CoA product.

Binding assays were conducted to detect binding to the BC domain, andthus, enabled identification of test compounds. Methods of conductingbinding assays are well known in the art. Direct measurement of thebinding of radiolabeled ligands is typically performed by incubatingnumerous concentrations of radioligand with a constant amount of targetpeptide under equilibrium binding conditions followed by determining theamount of labeled probe specifically bound. “Specifically bound” isdefined as total binding minus non-specific binding, where non-specificbinding is determined in the presence of excess unlabeled ligand. Thestrength of the binding interaction between the BC domain and soraphenis high and comparable to binding of soraphen to the full length ACCase(FIGS. 2, 3 and 4). The dissociation constant of the BC domaincomplexes, for example for soraphen, is from about 10⁻⁴ to about 10⁻¹⁴M, and preferably at 10⁻⁷ M to 10⁻¹⁴ M, and more preferably at least10⁻⁸ M to 10⁻¹⁴ M, and still more preferably lower than 2×10⁻⁹ M.Complexes can be formed by covalent or noncovalent interactions. Onceone possesses a radiolabeled ligand that binds to a target protein, anyadditional compound that is not radiolabeled can be assayed for bindingto the same site using a competition binding assay. A competitionbinding assay is performed by incubating a constant concentration oftarget protein and radiolabled ligand with numerous concentrations oftest compounds under equilibrium conditions followed by determining theamount of radiolabeled ligand that is specifically bound.

Thus, a further aspect of the present invention is a compositioncomprising: (a) an aqueous carrier solution; and (b) the peptide (orACCase BC domain described herein) solubilized in said aqueous carriersolution. The composition is useful for, among other things, the bindingor screening assays described herein. In general, “solubilized” meansthat the peptide is homogeneously or uniformly dissolved or dispersed inthe carrier solution in a manner that makes the peptide in thecomposition available for participation in the binding events (e.g.,soraphen binding) described herein. The carrier solution may be anysuitable aqueous solution that comprises, consists of or consistsessentially of water, along with other typical optional ingredients suchas buffers, agents for adjusting pH, preservatives, etc. In general, thepeptide is included in the composition in any suitable amount, forexample from 0.001, 0.01 or 0.1 nanograms up to 0.1, 1, 10, or 20milligrams per milliliter of aqueous carrier solution. The peptide is ina physical form in the composition that renders it suitable for abinding assay and thus has a soraphen dissociation constant in saidcomposition of, for example, from 10⁻⁴ Up to 10⁻¹⁴ M. The pH of thecomposition may be at, or adjusted to be at, a pH suitable for bindingstudies, such as a pH of 5 through 9. Preferably, the EvolutionaryChemistry process as referenced herein could be utilized to identifytest compounds that bind to the BC domain. In such instance, thecomposition should be comprised of an aqueous carrier solutioncontaining a BC domain peptide, possessing a soraphen dissociationconstant of 10⁻⁸ to 10⁻⁹ M. The BC domain would be utilized at aconcentration of 0.02 to 20 milligrams per milliliter at pH 7 incubatedin combination with one or more RNA-tethered test compounds for 1 hourto enable equilibrium binding to occur. Depending upon the level ofstringency applied, low affinity test compounds would be washed awayfrom the BC domain peptide and high affinity binding compounds would beretained. The retained compounds are potential ACCase inhibitors.

Alternatively, ACCase inhibitors could be identified in a screen basedon the principle of competition binding with soraphen. As mentionedpreviously, one way to detect competitive binding is by use ofradiolabeled soraphen. In such instance, the composition should becomprised of an aqueous carrier solution containing a BC domain peptide,possessing a soraphen dissociation constant of 10⁻⁸ to 10⁻⁹ M. The BCdomain would be utilized at a concentration of 5 nM to 10 nM at pH 7incubated in combination with ³H-soraphen (with a specific activitygreater than 500 cpm per picomole) at a concentration 10% to 90% that ofthe BC domain, and with one or more test compounds at a concentration of10⁻⁴ M to 10⁻¹⁰ M for 1 hour or more to enable equilibrium binding. Theamount of ³H-soraphen that remains bound to the BC domain would then bedetermined. A reduction in the amount of bound soraphen indicates thatthe test compound or compounds can bind to the same site on the BCdomain and thus represent potential ACCase inhibitors.

Another preferred method to screen for ACCase inhibitors based on theprinciple of competitive binding with soraphen is by a fluorescencepolarization assay. In this method, a fluorescent soraphen derivativethat retained high affinity for BC domains would need to be acquired orprepared through standard synthetic procedures. In such instance, thecomposition should be comprised of an aqueous carrier solutioncontaining a BC domain peptide, possessing a fluorescent-soraphenderivative dissociation constant of 10⁻⁶ M to 10⁻⁹ M. The BC domainwould be utilized at a concentration approximately equal to thedissociation constant of the fluorescent probe, and incubated in thepresence of one or more test compounds at a concentration of 10⁻⁴ M to10⁻¹⁰ M for 1 hour or more to enable equilibrium binding. Thefluorescence polarization would then be measured. Since the fluorescencepolarization is directly related to the amount of fluorescent-soraphenderivative bound, a reduction in fluorescence polarization indicatesthat the test compound or compounds can bind to the same site on the BCdomain and thus represent potential ACCase inhibitors.

Evolutionary chemistry (EC) as described herein relates to the processwherein product libraries are formed by combining a pool of firstchemical reactants coupled to a nucleic acid with a pool of freechemical reactants. The coupled nucleic acid is capable of mediating thechemical reaction which leads to the product library and further thenucleic acid is amplifiable so a product which has a predetermineddesirable characteristic can be enriched for and identified from theproduct library. In its most general form, a nucleic acid-reactant testmixture is formed by attaching a first reactant (R) to each of thenucleic acids in a test mixture (containing 10² to 10¹⁸ nucleic acidswith randomized sequences). The nucleic acid-reactant test mixture istreated with other free reactants that will combine with the firstreactant (R) to form different products. It is important to note thatfrom the nucleic acid test mixture (NA), discrete nucleic acid sequenceswill be associated with facilitating the formation of the differentshaped products and are denoted, for example, by sequence-A, sequence-Band sequence-C. The products may differ in shape, reactivity or bothshape and reactivity. Partitioning of the desirable product shape orreactivity is accomplished by binding to or reaction with a target.Proteins, small molecules, lipids, saccharides, etc., are all examplesof targets (T). After binding to or reacting with the target thenon-interacting products, which are attached to sequence-B andsequence-C are separated from sequence-A and discarded. The nucleic acidsequence-A is then amplified by a variety of methods known to thoseexperienced in the art. Sequence-A is then used to facilitate theassembly of the desirable product by facilitating the specific reactionto form the selected product on treatment with the mixture of startingreactants. In a typical reaction, Sequence-A can be reattached to thefirst reactant, however, said reattachment is not always required. Thisis an idealized case and in many examples the nucleic acid facilitatormay assemble more than one product from the starting mixture, but all ofthe products selected will have the desired properties of binding to orchemical reaction with the target. EC is more fully described in U.S.Pat. Nos. 6,048,698; 6,030,776; 5,858,660; 5,789,160; 5,723,592; and5,723,289.

In sum, BC peptide domains, as exemplified by Ustilago pCS8, areexpressed at high levels, can be purified to homogeneity, are stableunder typical laboratory conditions, and exhibit high affinity soraphenbinding comparable to that of full length ACCase (FIG. 5). Therefore, itis an excellent agent for use in target based affinity binding screensand selections, including but not limited to evolutionary chemistryselections, for the identification of ACCase inhibitors.

In some assays it may be desirable to use a first peptide of the presentinvention in conjunction (e.g., sequentially or simultaneously) with asecond peptide that serves as a counterselection agent. For oneembodiment, the counterselection agent may be a peptide of the samespecies as the first peptide (that is, with substantially the same aminoacid sequence as the first peptide), but with a nonfunctional BC domain(for example, by introduction of a deletion or substitution mutationtherein), to select against agents that bind non-specifically (e.g., notat the soraphen binding site) to the first peptide. An example would bean S. cerevisiae first peptide and a corresponding S. cerevisiae secondpeptide in which the second peptide contains a mutation that disruptssoraphen binding (e.g., S77->Y). In another embodiment, the secondpeptide counterselection agent may be a peptide of a different speciesas the first peptide, but with a functional BC domain, to detect agentsthat bind to and act on the first species but not the second species.For example, the first peptide may be non-mammalian, and the secondpeptide may be ammalian or human (e.g., to select against agents thatare active on the mammalian or human ACCase). Where a species containstwo different ACCases such as does human, the first and second peptidemay be of the same species but a different ACCase (e.g., human ACC1 andhuman ACC2). In either embodiment, the first and second peptides can beprovided together as kits or sets, either per se or ascompositions/formulations as described above, which may be stored,utilized and/or packaged together, optionally including instructions fortheir use in assays as described herein.

While the present invention has been described primarily with referenceto a ACCase BC domains isolated from Ustilago maydis (FIG. 6; SEQ ID NO:2), Phytophthora infestans (FIG. 7; SEQ ID NO: 4), Magnaporthe grisea(SEQ ID NO: 6), Saccharomyces cerevisiae (SEQ ID NO: 8), and Homosapiens (SEQ ID NOS: 10, 12, 14, 16), it will be appreciated thatdistantly related organisms may be substituted for the organismsdescribed herein. For example, peptides of the present invention may beisolated from other fungal, insect, or plant species, such as set forthin Tables 1-3 below, including any other members of the kingdoms,divisions, classes, orders or families set forth therein, as well asnematodes and mammals.

TABLE 1 Fungal Pests (Kingdom = Fungi if Division is not Oomycota; IfDivision = Oomycota, then Kingdom = Chromista) Common Major GenusSpecies Name Family Order Class Division Crop Magnaporthe grisea riceblast Magnaporthaceae Diaporthales Ascomycetes Ascomycota rice Erysiphegraminis powdery Erysiphaceae Erysiphales Ascomycetes Ascomycota wheattritici mildew Septoria nodorum septoria Leptosphaeriaceae PleosporalesAscomycetes Ascomycota wheat (Leptosphaeria) and tritici Gaeumannomycesgraminis take-all Pythiaceae Pythiales Oomycetes Oomycota wheat Pythiumspp Pythiaceae Pythiales Oomycetes Oomycota turf Puccinia sorghi stalkrot/rust Pucciniaceae Uredinales Urediniomycetes Basidiomycota maizeAspergillus flavus Trichocomaceae Eurotiales Ascomycetes Ascomycotamaize Phytophthora Infestans late blight Pythiaceae Pythiales OomycetesOomycota potatoes Fusarium spp wilt Nectriaceae Hypocreales AscomycetesAscomycota potatoes Botrytis spp Clavicipitaceae Hypocreales AscomycetesAscomycota tree/vines Alternaria spp Pleosporaceae PleosporalesAscomycetes Ascomycota Cercospora spp MycosphaerellaceaeMycosphaerellales Ascomycetes Ascomycota Rhizoctonia spp PlatygloeaceaePlatygloeales Ustilaginomycetes Basidiomycota CeratobasidiaceaeCeratobasidiales Basidiomycetes Basidiomycota Peronospora spp DownyPeronosporaceae Peronosporales Oomycetes Oomycota mildew ColletotrichumGlomerella Glomerellaceae Ascomycetes Ascomycota spp Bremia spp DownyPeronosporaceae Peronosporales Oomycetes Oomycota mildew

TABLE 2 Insect Pests (Kingdom = Animalia; Phylum = Arthropoda) MajorGenus Species Common Name Family Order Class Crop Nilaparvata lugensBrown planthopper Delphacidae Hemiptera Insecta rice Mayetioladestructor Hessian fly Cecidomyiidae Diptera Insecta wheat Heliothis zeaCorn earworm/ Noctuidae Lepidoptera Insecta maize bollworm Ostrinianubilalis European cornborer Pyralidae/Crambidae Lepidoptera Insectamaize Diabrotica spp Corn rootworm Chrysomelidae Coleoptera Insectamaize Myzus spp aphid Aphididae Homoptera Insecta potato Leptinotarsadecemlineata Colorado beetle Chrysomelidae Coleoptera Insecta potatoPectinophora gossypiella Pink bollworm Gelechiidae Lepidoptera Insectacotton Heliothis spp Noctuidae Lepidoptera Insecta cotton whitefliesAleyrodidae Homoptera Insecta Potato leafhopper Cicadellidae HemipteraInsecta Plutella xylostella Diamondback moth Plutellidae LepidopteraInsecta Chaetocnema Flea beetles Chrysomelidae Coleoptera Insecta spp

TABLE 3 Weedy Pests (Kingdom = Plantae; Division = Magnoliophyta) MajorGenus Species Common Name Family Order Class Crop Echinochloa crus-galliBarnyard grass Poaceae Cyperales Liliopsida rice, cotton Echinochloacolonum Poaceae Cyperales Liliopsida maize Avena fatua Wild oats PoaceaeCyperales Liliopsida wheat Polygonum convolvulus black bindweedPolygonaceae Polygonales Magnoliopsida wheat Cyperus rotundus sedgeCyperaceae Cyperales Liliopsida maize, cotton Chenopodium albumlambsquarters Chenopodiaceae Caryophyllales Magnoliopsida potato Galiumspp bedstraw Rubiaceae Ipomoea spp morningglory Convolvulaceae SolanalesMagnoliopsida Amaranthus spp pigweed Amaranthaceae CaryophyllalesMagnoliopsida Digitaria spp Crabgrass Poaceae Cyperales LiliopsidaLolium spp ryegrass Poaceae Cyperales Liliopsida Sorghum halepenseJohnson grass Poaceae Cyperales Liliopsida Panicum miliaceum Wild prosoPoaceae Cyperales Liliopsida millet Senna spp Velvet leaf FabaceaeFabales Magnoliopsida

The present invention is explained in greater detail in the followingnon-limiting Examples and the Figures herein, in which the followingabbreviations are used: pCS8—Ustilago maydis, basidomycete, N-terminalHis-tag BC domain, 64.6 kDa protein; pCS11—Ustilago maydis,basidomycete, full length ACCase with C-terminal His-tag, 241.4 kDaprotein; pCS15−Phytophthora infestans, oomycete C-terminal His-tag BCdomain, 63.3 kDa protein; pCS16—Saccharomyces cerevisiae, wild typeN-terminal His-tag BC domain, 68.0 kDa protein; pCS16M—Saccharomycescerevisiae, S77Y mutant, N-terminal His-tag BC domain, 68.0 kDa protein;pCS17−Magnaporthe grisea, ascomycete, N-terminal His-tag BC domain, 68.2kDa protein; pCS19—Homo sapiens, C-term His-tag ACC1 BC domain, 71.2 kDaprotein; pCS20—Homo sapiens, C-term His-tag ACC2 BC domain, 86.7 kDaprotein; pCS201−Escherichia coli, N-terminal His-tag BC protein, 51.6kDa protein; pCS204—Saccharomyces cerevisiae, C-terminal His-tag wildtype full length ACCase protein, 254.3 kDa protein; andpCS204M—Saccharomyces cerevisiae, C-terminal His-tag S77Y mutant fulllength ACCase protein, 254.3 kDa protein.

Example 1

A. Preparation of BC Domain Peptide. E. coli cultures transformed withprotein expression constructs for BC domains with either N or C terminalhis-tags (as illustrated in FIG. 5 for pCS8) were induced by theaddition of IPTG (0.2 mM) at an OD₆₀₀=0.5. The cultures were grownovernight at 18° C., harvested and stored at −80° C. The bacterialpellet was resuspended in a buffer containing 50 mM NaH₂PO₄ (pH8), 300mM NaCl, 10 mM imidazole, protease inhibitor and 1 mg/mL lysozyme. Thelysate was sonicated and nuclease was added. The lysate was thenincubated with Ni-NTA resin (Novagen) for 1 hour at 4° C. pCS8 waseluted with a buffer containing 50 mM NaH₂PO₄ (pH8), 300 mM NaCl, and250 mM imidazole. Fractions containing pCS8 were combined and ammoniumsulfate precipitated (40% ammonium sulfate). The pellet from theammonium sulfate precipitation was resuspended in SB (SB=200 mM NaH₂PO₄pH 7.0, 10% glycerol). Protein concentrations were determined byBradford analysis. The purified protein was stored at −80° C.

B. Purification of BC Domain Peptide. A one-step purification ofhis-tagged BC domain peptides, as exemplified for pCS8, onNi-NTA-agarose yields protein that is approximately 90-95% pure asjudged by SDS-PAGE was utilized. This method is similar to thepurification performed with a histidine-tag attached to the aminoterminus of a mutant form of the enzyme and nickel affinitychromatography as described in C. Blanchard et al., “Mutations at FourActive Site Residues of Biotin Carboxylase Abolish Substrate-InducedSynergism by Biotin,” Biochemistry, vol. 38, pp. 3393-3400 (1999). Afterelution from the Ni-NTA agarose column, the pCS8 protein wasprecipitated by adding an equal volume of saturated ammonium sulfate.The precipitated protein was then resuspended in SB to a concentrationof 10 to 20 milligrams per milliliter and stored at −80° C. until used.This method is utilized for purification of all BC domains describedherein. Because purer preparations may be required for some purposes, anadditional polishing step was investigated. For this purpose, a singleUNO-Q (Bio-Rad) anion exchange step subsequent to the Ni-NTA-agarosechromatography purified pCS8 to apparent homogeneity with good yield.UNO-Q is a fast flow matrix that is readily amenable to scale-up. Seealso FIG. 8.

Example 2

ACCase Activity Assays. The following methods were employed to detectACCase activity. See also FIGS. 9A and 9B.

Method 1: Assay-Spectrophotometric. ACCase activity was measuredspectrophotometrically by coupling the production of ADP to theoxidation of NADH using pyruvate kinase and lactate dehydrogenase. Thisassay was used to measure either overall ACCase activity by supplyingacetyl CoA as a substrate, or BC activity by supplying free biotin as asubstrate (note, however, that this has only been demonstrated with“prokaryotic type” BC's). This assay is best suited for purifiedprotein. This assay would be used to test for enzymatic activity ofcompounds identified by virtue of their binding to isolated BC domains,including but not limited to the EC process.

To establish this assay, E. coli biotin carboxylase was first cloned andexpressed according to the literature (Biochemistry 38:3393-3400, 1999).As seen in FIG. 9A, the activity of E. coli BC towards a free biotinsubstrate was readily detectable. Under the conditions of the assay,activity was detected with as little as 40 ng protein, and a maximalvelocity was reached between 2 and 20 μg pCS201. In repeated attemptswith multiple pCS8 preparations, however, no activity was detected usingup to 7 μg protein. We conclude that pCS8 is unable to carboxylate freebiotin, as would be expected since such activity has not been detectedutilizing eukaryotic BC domains.

To measure overall ACCase activity, a full-length Ustilago maydis ACCasewas cloned, expressed in E. coli with a C-terminal His-tag (pCS11), andpurified. As seen in FIG. 9B(i), the time dependent oxidation of NADH,detected by a decrease in absorbance at 340 nm, was dependent on bothpCS11 protein as well as acetyl CoA substrate.

Method 2: Assay-14C Isotope Exchange. ACCase catalyzes the formation ofmalonyl CoA from acetyl-CoA and bicarbonate. The isotope exchange assayis designed to monitor the incorporation of ¹⁴C from bicarbonate intothe malonyl CoA product. Malonyl CoA is acid and heat stable so theunreacted H¹⁴CO₃ ⁻ can be removed by acidification followed byevaporation. As can be seen in FIG. 9B(ii) and consistent with thespectrophotometric assay, activity (measured as the incorporation of ¹⁴Cinto malonyl CoA) was dependent on both pCS11 protein and acetyl CoAsubstrate. Because this an endpoint assay, it is less suitable than thespectrophotometric assay for kinetic measurements; however, it issuperior for detecting activity in crude preparations. It will also beused to test for enzymatic activity of compounds identified by virtue oftheir binding to isolated BC domains, including but not limited to theEC process.

Example 3

Soraphen binding assay. Affinity based screens and selection assays,including but not limited to EC selections, rely on binding of smallmolecules and not inhibition of enzymatic activity. Therefore, despitethe lack of enzymatic activity, pCS8 would be a suitable affinity basedscreening or selection agent if it retained the high affinity soraphenbinding activity of the full-length Ustilago ACCase. Soraphen A wastritium labeled by Sibtech, Inc. (Newington, Conn.) and used for bindingexperiments with pCS8 protein. Briefly, 6.7 nM purified pCS8 protein wasincubated with various concentrations (approximately 0.5-20 nM) of³H-soraphen in PNT buffer (100 mM NaH₂PO₄, 150 mM NaCl, 0.01% TritonX-100, pH 7.0) for 45 min at room temperature. pCS8 protein (with boundligand) was separated from free ligand on NAP-5 desalting columns(Amersham Biosciences) and the amount of bound ³H-soraphen determined byliquid scintillation counting. Non-specific binding was determined withduplicate samples containing 2 μM cold soraphen. The data were fit bynon-linear regression to a one site ligand binding equation:Y=B_(max)*X/(K_(D)+X) where Y=bound ligand and X=free ligand.

pCS8 exhibited saturable binding of ³H-Soraphen consistent with a singlehigh affinity binding site (FIG. 3). The data shown for pCS8 arecombined from two experiments with independent protein prepsdemonstrating little prep-to-prep variability. A negative control wasprovided by pCS201. pCS201 encodes an N-terminal His-taggedenzymatically active E. coli BC that is not inhibited by soraphen. Asexpected, pCS201 did not exhibit high affinity soraphen binding.

Non-linear regression fits of the data gave an estimate of 1.5 nM forthe K_(D) Of the soraphen-pCS8 interaction (FIG. 4). This is in goodagreement with the K_(D) estimate of the soraphen-pCS11 full lengthACCase of 1.6 nM (FIG. 2) and is in good agreement with the publishedvalue of a 1.4 nM K_(i) for soraphen inhibition of Ustilago ACCaseactivity (Heike Behrbohm Ph.D. thesis, Braunschweig Techn. Univ., 1996).As such, the pCS8 is a suitable affinity-based screening and selectionagent as it retains high-affinity soraphen binding comparable tofull-length Ustilago ACCase.

Example 4

Additional characterization of pCS8. No protein degradation or loss ofsoraphen binding was seen after incubation of SB solubilized pCS8peptide for 24 h at room temperature. No protein degradation or loss ofsoraphen binding was seen after storage of SB solubilized pCS8 peptidefor 5 weeks at −80° C., including multiple freeze thaws.

Example 5

Partial summary of BC domains generated. A number of biotin carboxylase(BC) domains that have been characterized herein and can be purified insufficient quantities for use in affinity based screens or selections,including but not limited to selections using Evolutionary Chemistry,and five BC domains are as follows: wild type versions from Ustilagomaydis, Phytophthora infestans, Magnaporthe grisea, and Saccharomycescerevisiae; and the mutated version from Saccharomyces cerevisiae. Aminoacid sequence alignments of the expressed domains are shown in FIG. 10.Identical residues are indicated with an asterisk, and the S to Ymutation in S. cerevisiae that abolishes soraphen binding is indicatedin bold.

Example 6

Generation of the Magnaporthe grisea BC domain. A preliminary sequenceof the entire genome of Magnaporthe grisea (ascomycete, causal agent ofrice blast) was recently released into the public domain by theWhitehead Institute in collaboration with Ralph Dean's lab at NorthCarolina State University. The full-length M. grisea ACCase gene was PCRamplified from genomic DNA, cloned, and sequenced. One small predictedintron was removed to create a full-length cDNA. The biotin carboxylasedomain was subcloned (based on alignment with our pCS8 U. maydis BCdomain) and inserted into a pET vector (5′His tag) to make pCS17. pCS17was expressed in E. coli and the His-tagged BC domain was purified andassayed for soraphen binding. As expected, the Magnaporthe BC domainexhibited high affinity soraphen binding, as demonstrated in FIG. 11.

Example 7

Cloning and expression of human ACCase genes. There are at least twoforms of the acetyl-CoA carboxylase enzyme in humans. ACC1 is acytosolic enzyme present at high levels in liver and lipogenic tissues,and is the primary species responsible for fatty acid synthesis. ACC2 isa mitochondrial enzyme found primarily in heart and muscle tissue, andis thought to regulate fatty acid oxidation. Biotin carboxylase domainsfrom human ACCases could potentially be useful as counterselectionagents with potential to select against mammalian toxicity.Additionally, agonists and inhibitors of human ACCase that candistinguish between ACC1 and ACC2 BC domains may have potentialpharmaceutical applications.

Complete genomic DNA sequences are available for both genes. Their BCdomains (based on homology with our pCS8 ustilago clone) weresuccessfully cloned by amplifying small exons from genomic DNA and usingPCR to splice them together. The ACC1 BC domain consists of 14 exons,104 bp to 237 bp in length, which were assembled to make an 1896 bp (SEQID NO: 9) BC domain cDNA. The ACC2 BC domain consists of 14 exons,108-661 bp in length, assembled to make a 2322 bp (SEQ ID NO: 11) BCdomain cDNA. Both BC domains were cloned into pET30 to make 3′His-tagged fusion proteins, The ACC1 clone is designated pCS19 andshould produce a fusion protein of 71.2 kD. The ACC2 clone is designatedpCS20 and should produce a fusion protein of 86.7 kD. Expressionanalysis showed that the ACC1 fusion protein is expressed at low levelsin E. coli and can be purified from the soluble fraction. The purifiedpCS19 protein exhibited high affinity soraphen binding (see FIG. 12).Alignments of the Ustilago and human ACCase domains are depicted in FIG.13. Besides being useful in identifying selective agrochemicals, aparticularly intriguing use of the human BC domains is to identifyspecific inhibitors that preferentially target the ACC2 domain, but notthe ACC1 domain, since such inhibitors could prove useful in controllingbody weight.

Example 8

This example demonstrates that pCS8 has binding characteristics amenablefor use in affinity based screening or selection procedures.

Soraphen off-rate determination. Kinetic aspects of binding interactionsare an important parameter in designing optimal conditions foraffinity-based screening and selection assays, including but not limitedto EC. Therefore, soraphen dissociation experiments were performed todetermine the off-rate for the binding interaction using the Ustilago BCdomain (pCS8). 53.6 pmol pCS8 protein was incubated with 30 pmol³H-soraphen in a volume of 2 ml for 15 min. The protein with boundsoraphen was separated from free soraphen on NAP5 columns and eluted ina total of 4 ml. Cold soraphen was added to 2 μM and dissociation of thebound soraphen was followed by removing 0.5 ml aliquots at various timesand applying them to NAP 5 columns to again separate bound from freeradiolabel. The 3H-soraphen in the eluted fractions was quantified byliquid scintillation counting and the data were fit to the followingequation: Y=Y_(max)*e^(−kt)+NS (FIG. 14).

This off rate corresponds to 10.7 min half-life. Note that the on-ratecan be calculated from this data since K_(d)=k_(off)/k_(on); sok_(on)=9.31×10⁵ M⁻¹ s⁻¹. At 10 μM protein, even with very low ligandconcentrations, such a binding interaction would approach equilibriumvery rapidly.

Example 9

Competitive Binding Assay. Isolated biotin carboxylase domains andradiolabeled soraphen can be employed in a competitive binding assay totest the ability of any compound to bind to the soraphen binding site.Like soraphen, such compounds are likely to inhibit ACCase activity. Toexemplify this assay, we prepared two soraphen derivatives withmodifications at the 5-position (soraphen A-conjugate) or 11-position(soraphen C-conjugate). Both derivatives were then tested in competitionbinding assays with both pCS8 and pCS11 proteins. The proteins wereincubated with ³H-soraphen and various concentrations of the 2conjugates for 1 hr. Bound ³H-soraphen was then separated from unboundon NAP 5 columns and quantified by scintillation counting. To estimatethe conjugates K_(i)'s, the resulting data was fit to an equation forheterologous competitive binding with ligand depletion (H. J. Motulsky,Analyzing Data with GraphPad Prism, 1999, GraphPad Software Inc., SanDiego Calif., www.graphpad.com):

$Y = {\frac{\lbrack{free}\rbrack*B\; \max}{\lbrack{free}\rbrack + {{Kd}\left( {1 + {\lbrack{cold}\rbrack/{Ki}}} \right)}} + {NS}}$

Representative data are shown in FIG. 15, in which the soraphenA-conjugate is labeled A-conj and the soraphen C-conjugate is labeledC-conj. As expected, there was no significant difference between theaffinities of the conjugates for the BC domain (pCS8) (FIG. 15A) and thefull-length protein (pCS11) (FIG. 15B). As a positive control for thisassay, an experiment using cold soraphen A as the competitor wasperformed and yielded a K_(i) estimate of 1.1 nM (data not shown),consistent with direct saturation binding experiments.

Example 10

Soraphen resistant Saccharomyces cerevisiae ACCase and ACCase BC domainmutants—Counterselection Agents. Mutation of Serine 77 to Tyrosine inthe S. cerevisiae ACCase protein has been shown to confer soraphenresistance (European Patent Application 94810710, 1994; and U.S. Pat.No. 5,641,666, 1997).

This soraphen resistance mutation was introduced into pCS204, a yeastexpression vector containing the full-length S. cerevisiae ACCase geneconstructed by cloning a PCR-amplified full-length S. cerevisiae ACCasegene into the expression vector pYES2 for inducible overexpression ofHis-tagged ACCase in S. cerevisiae. The resulting construct wasdesignated pCS204M. The S. cerevisiae biotin carboxylase domains frompCS204 and pCS204M were then subcloned into an E. coli pET expressionvector to form pCS16 and pCS16M, respectively. Like pCS8, both of theseconstructs express their respective BC domains as N-terminal-His-taggedfusion proteins to facilitate purification. Expression analysisdemonstrated that both pCS16 and pCS 16M yielded comparable amounts ofpurified, soluble protein. The products were then analyzed by sizeexclusion chromatography and, like pCS8 protein, both were found toexist primarily as monomers (>90%). The proteins were then tested forsoraphen binding and the results are shown in FIG. 16. pCS16 proteinexhibited high-affinity soraphen binding comparable to pCS8 protein. Incontrast, soraphen binding by pCS16M was similar to the non-specificcontrol (Data not shown) confinning that introduction of this singleamino acid mutation into a BC domain abolishes soraphen binding.Therefore, pCS16M protein would be an excellent counter-selection agentto eliminate non-soraphen-binding-site interactions.

The effect of the mutation on full-length ACCase was also assessed.pCS204 and pCS204M were overexpressed in S. cerevisiae and purified byNi-NTA chromatography. Both proteins appeared identical on SDS-PAGE(Data not shown).

The proteins were then assayed for ACCase activity using the ¹⁴C isotopeexchange assay. The resulting data were similar to those from apublished report (Curr. Genet. 25:95-100 (1994)) comparing the activityof the endogenous protein from wild type and mutant yeast, anddemonstrate that pCS204M protein activity is insensitive to soraphen butstill sensitive to avidin inhibition (Table 4).

TABLE 4 Relative Enzymatic Activity (%) Mutant Treatment WT enzyme*enzyme* pCS204 pCS204M Control 100.0 100.0 100.0 100.0 +150 μg/mL 0.874.7 7.5 105 soraphen A +1.5 μg/mL 1.0 74.9 11.6 101 soraphen A +1.5ng/mL 71.8 79.4 92.0 103 soraphen A +250 μg/ml avidin 0.4 0.1 2.1 2.5 -acetyl CoA 0.6 0.2 2.0 2.8 *Data from Curr. Genet. 25: 95-100 (1994).

Finally, pCS204 and pCS204M were assayed for soraphen A binding and theresults are shown in FIG. 17. Full-length pCS204 protein bound soraphenwith similar high affinity as the BC domain expressed by pCS16. Soraphenbinding by pCS204M, like that of pCS 16M, was comparable to thenon-specific control.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A peptide comprising an Acetyl CoA carboxylase (ACCase) having adeleted biotin binding domain, having a deleted carboxy transferasedomain, and having a functional biotin carboxylase domain.
 2. Thepeptide according to claim 1, wherein said ACCase is selected from thegroup consisting of mammal, insect, yeast, Ascomycota, Basidiomycota,and Oomycota ACCase.
 3. The peptide according to claim 1, wherein saidcarboxylase is Ustilago maydis carboxylase.
 4. The peptide according toclaim 1, wherein said carboxylase is Phytopthora infestans carboxylase.5. The peptide according to claim 1, wherein said carboxylase isMagnaporthe grisea carboxylase.
 6. The peptide according to claim 1,wherein said carboxylase is Saccaromyces cerevisiae carboxylase.
 7. Thepeptide according to claim 1, wherein said carboxylase is humancarboxylase.
 8. The peptide according to claim 1 having the amino acidsequence given in SEQ ID NO:
 2. 9. The peptide according to claim 1selected from the group consisting of peptides having an amino acidsequence as given in SEQ ID NO; 4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17through SEQ ID NO:
 71. 10. The peptide according to claim 1, whereinsaid peptide is a monomer.
 11. The peptide according to claim 1, whereinsaid peptide binds to soraphen.
 12. The peptide according to claim 1,wherein said peptide binds to soraphen and has a soraphen dissociationconstant of from 10⁻⁷ to 10⁻¹⁴ M.
 13. A composition comprising: (a) anaqueous carrier solution; and (b) the peptide of claim 1 solubilized insaid aqueous carrier solution; with said peptide included in saidcomposition in an amount of from 0.001 nanograms to 20 milligrams permilliliter of aqueous carrier solution; said peptide having a soraphendissociation constant in said composition of from 10⁻⁷ to 10⁻¹⁴ M; andsaid composition having a pH of from 5 through
 9. 14. A nucleic acidthat encodes a peptide according to claim
 1. 15. A recombinant host cellthat contains a nucleic acid according to claim 14 and expresses theencoded peptide.
 16. A method of identifying Acetyl CoA carboxylaseinhibitors or activators, comprising: a) combining a peptide accordingto claim 1 and a compound to be tested for the ability to bind to saidbiotin carboxylase domain, under conditions that permit binding to saidbiotin carboxylase domain; b) determining whether or not said compoundbinds to said biotin carboxylase domain, the presence of bindingindicating said compound is or may be an Acetyl CoA carboxylaseinhibitor or activator.
 17. The method of claim 16, further comprisingthe steps of: c) employing a compound identified as binding in step (b)in an assay to detect inhibition or enhancement of Acetyl CoAcarboxylase activity; and d) selecting a compound identified in step (c)that inhibits or activates Acetyl CoA carboxylase activity.
 18. A methodof identifying fungicides, comprising: a) combining a peptide accordingto claim 1 and a compound to be tested for the ability to bind to saidbiotin carboxylase domain, under conditions that permit binding to saidbiotin carboxylase domain; b) determining whether or not said compoundbinds to said biotin carboxylase domain, the presence of bindingindicating said compound is or may be a fungicide; c) employing acompound identified as binding in step (b) in an assay to detectinhibition of Acetyl CoA carboxylase activity; and d) selecting acompound identified in step (c) that inhibits Acetyl CoA carboxylaseactivity.
 19. A kit comprising: (a) a first peptide of claim 1; incombination with (b) a second peptide of claim 1, wherein said first andsecond peptides are from different species.
 20. A kit of claim 19,wherein said first peptide is a non-mammalian peptide and said secondpeptide is a mammalian peptide.
 21. A kit comprising: (a) a firstpeptide of claim 1; in combination with (b) a second peptide comprisingan ACCase having a deleted biotin binding domain, having a deletedcarboxy transferase domain, and having a non-functionalbiotin-carboxylase domain; wherein said first and second peptide arefrom the same species.
 22. A kit of claim 19, wherein said first andsecond peptide are both S. cerivasae peptides.